Crystalline 1alpha-hydroxyvitamin D2 and method of purification thereof

ABSTRACT

A method of purifying 1α-hydroxyvitamin D 2  to obtain 1α-hydroxyvitamin D 2  in crystalline form. The method includes the steps of boiling a solvent selected from the group consisting of ethyl formate, ethyl acetate and a 2-propanol-hexane mixture under inert atmosphere, dissolving a product containing 1α-hydroxyvitamin D 2  to be purified in the solvent, cooling the solvent and dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of 1α-hydroxyvitamin D 2  crystals, and recovering the 1α-hydroxyvitamin D 2  crystals. Petroleum ether is also added to the solvent after dissolving the product to be purified in the solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Patentapplication Ser. No. 09/233,738 filed Jan. 20, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with United States Government supportawarded by the National Institutes of Health (NIH), Grant #DK-14881. TheUnited States Government has certain rights in this invention.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] The present invention relates to purification of organiccompounds, and more particularly to the purification of1α-hydroxyvitamin D₂ (1α-OH-D₂) by preparing it in crystalline form.

[0004] Purification of organic compounds, especially those designatedfor pharmaceutical use, is of considerable importance for chemistssynthesizing such compounds. Preparation of the compound usuallyrequires many synthetic steps and, therefore, the final product can becontaminated not only with side-products derived from the last syntheticstep of the procedure but also with compounds that were formed inprevious steps. Even chromatographic purification, which is a veryefficient but relatively time-consuming process, does not usuallyprovide compounds which are sufficiently pure to be used as drugs.

[0005] Depending on the method used to synthesize 1α-hydroxyvitamin Dcompounds, different minor undesirable compounds can accompany the finalproduct. Thus, for example, if direct C-1 hydroxylation of 5,6-transgeometric isomer of vitamin D is performed, followed by SeO₂/NMOoxidation and photochemical irradiation [see Andrews et al., J. Org.Chem. 51, 1635 (1986); Calverley et al., Tetrahedron 43, 4609 (1987);Choudry et al., J. Org. Chem. 58, 1496 (1993)], the final1α-hydroxyvitamin D product can be contaminated with 1α-hydroxy- as wellas 5,6-trans isomers. If the method consists of C-1 allylic oxidation ofthe 4-phenyl-1,2,4-triazoline-3,5-dione adduct of the previtamin Dcompound, followed by cycloreversion of the modified adduct under basicconditions [Nevinckx et al., Tetrahedron 47, 9419 (1991); Vanmaele etal., Tetrahedron 41, 141 (1985) and 40, 1179 (1991); Vanmaele et al.,Tetrahedron Lett. 23, 995 (1982)], one can expect that the desired1α-hydroxyvitamin can be contaminated with the previtamin5(10),6,8-triene and 1 P-hydroxy isomer. One of the most useful C-1hydroxylation methods, of very broad scope and numerous applications, isthe experimentally simple procedure elaborated by Paaren et al. [see JOrg. Chem. 45, 3253 (1980) and Proc. Natl. Acad. Sci. U.S.A. 75, 2080(1978)]. This method consists of allylic oxidation of 3,5-cyclovitamin Dderivatives, readily obtained from the buffered solvolysis of vitamin Dtosylates, with SeO₂/ t-BuOOH and subsequent acid-catalyzedcycloreversion to the desired 1α-hydroxy compounds. Taking into accountthis synthetic path it is reasonable to assume that the final productcan be contaminated with 1β-hydroxy epimer, 5,6-trans isomer and theprevitamin D form. 1α-hydroxyvitamin D₄ is another undesirablecontaminant found in 1α-hydroxyvitamin D₂ synthesized from vitamin D₂ orfrom ergosterol. 1α-hydroxyvitamin D₄ results from C-1 oxidation ofvitamin D_(4,) which in turn is derived from contamination of thecommercial ergosterol material. Typically, the final product may containup to about 1.5% by weight 1α-hydroxyvitamin D_(4.) Thus, a purificationtechnique that would eliminate or substantially reduce the amount of1α-hydroxyvitamin D₄ in the final product to less than about 01.-0.2%would be highly desirable.

[0006] The vitamin D conjugated triene system is not only heat- andlight-sensitive but it is also prone to oxidation, leading to thecomplex mixture of very polar compounds. Oxidation usually happens whena vitamin D compound has been stored for a prolonged time. Other typesof processes that can lead to a partial decomposition of vitamin Dcompounds consist of the some water-elimination reactions; their drivingforce is allylic (1α-) and homoallylic (3β-) position of the hydroxygroups. The presence of such above-mentioned oxidation and eliminationproducts can be easily detected by thin-layer chromatography. Thus, forexample, using precoated aluminum silica sheets [with UV indicator; fromEM Science (Cherry Hill, N.J.)] and solvent system hexane-ethyl acetate(4:6), the spot of 1α-OH-D₂ (R_(f) 0.27) and its elimination products(R_(f)'s ca. 0.7-0.9) are visible in ultraviolet light. Also, afterspraying with sulfuric acid and heating, an additional spot can bevisualized (R_(f) 0), derived from oxidation products.

[0007] Usually, all 1α-hydroxylation procedures require at least onechromatographic purification. However, even chromatographically purified1α-hydroxyvitamin D₂, although showing consistent spectroscopic data,suggesting its homogeneity, does not meet the purity criteria requiredfor therapeutic agents that can be orally, parenterally or transdermallyadministered. Therefore, it was evident that a suitable method ofpurification of 1α-hydroxyvitamin D₂ is required.

[0008] Since it is well known that the simplest procedure that can beused for compound purification is a crystallization process, it wasdecided to investigate purification of 1α-OH-D₂ by means ofcrystallization. The solvent plays a crucial role in the crystallizationprocess, and is typically an individual liquid substance or a suitablemixture of different liquids. For crystallizing 1α-hydroxyvitamin D₂,the most appropriate solvent and/or solvent system is characterized bythe following factors:

[0009] (1) low toxicity;

[0010] (2) low boiling point;

[0011] (3) significant dependence of solubility properties with regardto temperature (condition necessary for providing satisfactorycrystallization yield); and

[0012] (4) relatively low cost.

[0013] It was found that highly apolar solvents (e.g. hydrocarbons) werenot suitable due to the low solubility of 1α-OH-D₂ in them. Quite thereverse situation occurred in highly polar solvent media (e.g.alcohols), in which 1α-OH-D₂ showed too high solubility. Therefore, itwas concluded that for the successful crystallization of 1α-OH-D₂, asolvent of medium polarity is required or, alternatively, a solventmixture consisting of two (or more) solvents differing considerably inpolarity. Interestingly, hexane, so frequently used for crystallizationpurposes with co-solvents like acetone, ethyl acetate or diethyl ether,was found less suitable for crystallization of 1α-OH-D₂. Unusually lowyields of crystallization were obtained when hexane-containing solventsystems were used. However, it was discovered that replacement of thehexane in such solvent mixtures with petroleum ether increasedsignificantly the yield of crystals. After numerous experiments it wasfound that an individual solvent, namely ethyl formate, was most usefulfor the crystallization of 1α-OH-D₂. In addition, binary and ternarysolvent systems namely: ethyl acetate-petroleum ether and2-propanol-hexane-petroleum ether, respectively, also performed well.These solvents are all characterized by low toxicity, and they are veryeasy to remove by evaporation or other well known methods. In all casesthe crystallization process occurred easily and efficiently; and theprecipitated crystals were sufficiently large to assure their recoveryby filtration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1a-1 h are graphs of ¹H NMR spectra (CDCl₃, 500 MHz) of thecrystals of 1α-hydroxyvitamin D₂ resulted after two crystallizationsusing the following solvent system: HCOOEt (FIGS. 1a and 1 b),AcOEt-petroleum ether (FIGS. 1c and 1 d) and iPrOH-hexane-petroleumether (FIGS. 1e and 1 f) as well as the spectrum of the solid1α-hydroxyvitamin D₂ material before crystallization (FIGS. 1g and 1 h);

[0015]FIGS. 2a-2 d are HPLC (10 mm×25 cm Zorbax-Sil column, 15%2-propanol in hexane, 4 mL/min; UV detection at 260 nm) profiles of thesolid 1α-hydroxyvitamin D₂ material before crystallization (FIG. 2a) andthe crystals resulted after two crystallizations using the followingsolvent system: HCOOEt (FIG. 2b), AcOEt-petroleum ether (FIG. 2c) andiPrOH-hexane-petroleum ether (FIG. 2d). In the region indicated by theasterisk (ca. 36 mL) sensitivity was decreased 20 times.

[0016]FIGS. 3a-3 i are HPLC (10 mm×25 cm Zorbax-Sil column, 15%2-propanol in hexane, 4 mL/min; UV detection at 260 nm) profiles of thecrystals of 1α-hydroxyvitamin D₂ resulted after single crystallizationusing the following solvent system: HCOOEt (FIG. 3a), AcOEt-petroleumether (FIG. 3d) and iPrOH-hexane-petroleum ether (FIG. 3g); the HPLCprofiles of mother liquors after single crystallization using thefollowing solvent systems: HCOOEt (FIG. 3b), AcOEt-petroleum ether (FIG.3e) and iPrOH-hexane-petroleum ether (FIG. 3h); and the HPLC profiles ofmother liquors after two crystallizations using the following solventsystem: HCOOEt (FIG. 3c), AcOEt-petroleum ether (FIG. 3f) andiPrOH-hexane-petroleum ether (FIG. 3i). Region with decreasedsensitivity (ca. 36 mL) is indicated by asterisk.

[0017]FIGS. 4a-4 f are Microscope-magnified images of the crystals of1α-hydroxyvitamin D₂ resulted after two crystallizations using thefollowing solvent system: HCOOEt (FIG. 4a -40x, FIG. 4b -100x),AcOEt-petroleum ether (FIG. 4c -100x, FIG. 4d -400x) andiPrOH-hexane-petroleum ether (FIG. 4e -100x, FIG. 4f -400x).

[0018]FIGS. 5a-5 d are HPLC (4.6 mm×25 cm Zorbax-Eclipse XDB-C18 column,7% water in methanol, 1.5 mL/min; UV detection at 260 nm) profiles ofthe solid 1α-hydroxyvitamin D₂ material before crystallization (FIG. 5a)and the crystals resulted after two crystallizations from: HCOOEt (FIG.5b), AcOEt-petroleum ether (FIG. 5c) and iPrOH-hexane-petroleum ether(FIG. 5d). In the region indicated by asterisk (ca. 15 min) sensitivitywas decreased 20 times.

[0019]FIGS. 6a and 6 b are illustrations of the three dimensionalstructure of 1α-hydroxyvitamin D₂ as defined by the atomic positionalparameters discovered and set forth herein.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a valuable method of purificationof 1α-hydroxyvitamin D₂, a pharmacologically important compound,characterized by the formula shown below:

[0021] The purification technique involves obtaining the1α-hydroxyvitamin D₂ product in crystalline form by utilizing acrystallization procedure wherein the 1α-hydroxyvitamin D₂ material tobe purified is dissolved using as the solvent or solvent system one ofthe following:

[0022] (1) a single solvent system, namely, ethyl formate;

[0023] (2) a binary solvent system, namely, ethyl acetate and petroleumether; or

[0024] (3) a ternary solvent system, namely, 2-propanol in combinationwith hexane and petroleum ether.

[0025] Thereafter, the solvent or solvent system can be removed byevaporation, with or without vacuum, or other means as is well known.The technique can be used to purify a wide range of final productscontaining 1α-hydroxyvitamin D₂ obtained from any known synthesisthereof, and in varying concentrations, i.e. from microgram amounts tokilogram amounts. As is well known to those skilled in this art, theamount of solvent utilized should be minimized and/or adjusted accordingto the amount of 1α-hydroxyvitamin D₂ to be purified.

[0026] The usefulness and advantages of the present crystallizationprocedures is shown in the following specific Examples. Solid1α-hydroxyvitamin D₂ product, obtained by Paaren's, supra, method,purified by flash chromatography on silica, and stored for few months inrefrigerator, was used as a suitable starting material. Although thismaterial still showed reasonably good 500 MHz ¹H NMR spectrum (FIGS. 1g,1 h), concomitant compounds were detected by straight- and reverse-phaseHPLC (FIGS. 2a and 5 a, respectively) and, moreover, the presence ofsome oxidation products was confirmed by TLC (presence of a spot atR_(f) 0). After recrystallization from the solvents listed above, theprecipitated material was observed under microscope to confirm itscrystalline form (FIGS. 4a-4 f). Additionally, in the case of crystalsprecipitated from ethyl formate, X-ray diffraction analysis wasperformed. The corresponding crops of crystals were then carefullyanalyzed and their significantly improved purity was confirmed bystraight-phase HPLC (FIGS. 2b, 2 c, 2 d), reverse-phase HPLC (FIGS. 5b,5 c, 5 d), TLC and 500 MHz ¹H NMR (FIG. 1a-1 f). Yields ofcrystallizations were high and the obtained crystals showed a relativelysharp melting point.

[0027] As it can be seen from FIG. 2b and 2 c, HPLC profiles of1α-hydroxyvitamin D₂ obtained after two crystallizations fromester-containing solvents indicate presence of a small amount of lesspolar impurities (peaks at R_(v) ca. 18 mL) which most likely originatefrom the corresponding 1α-OH-D₂ formate(s) and acetate(s) formed inminimal quantities in the equilibrium processes:

HCOOEt(CH3COOEt)+ROH

HCOOR (CH3COOR)+EtOH

[0028] However, the small amount of such esterificated compounds (lessthan 0.4%) presents no problem for pharmacological application of thecrystalline vitamin D₂ compound due to the well-known fact that vitaminD esters undergo hydrolysis in living organisms.

[0029] Also, the corresponding reverphase HPLC profiles of therecrystallized 1α-hydroxyvitamin D_(2,) shown in FIGS. 5b, 5 c and 5 d,clearly indicate a considerable improvement in the compound's purity.The important observation consists of the significantly diminishedproportion of the concomitant 1α-hydroxyvitamin D₄ (peak of retentiontime ca. 18 mL) in the recrystallized compound. The content of thisimpurity has decreased more than 4 times (4.1-4.3) in respect to itsvalue in the starting 1α-hydroxyvitamin D₂ product and does not exceed0.2%.

[0030] The described crystallization processes of the synthetic1α-hydroxyvitamin D₂ product represents a valuable purification method,which can remove not only some side products derived from the syntheticpath, but, moreover, concomitant 1α-hydroxyvitamin D₄. Such impurity isthe result of the contamination of natural ergosterol with its22,23-dihydro analog and, consequently, vitamin D₄ is present indifferent proportions in the commercially available vitamin D₂. Columnchromatography and straight-phase HPLC separation of 1α-hydroxyvitaminD₄ (formed after 1α-hydroxylation process) from 1α-hydroxyvitamin D₂ ispractically impossible due to their similar chromatographical propertiesand larger-scale separation is also difficult by reverse-phase HPLC.

CRYSTALLIZATION OF 1α-HYDROXYVITAMIN D₂ EXAMPLE 1

[0031] Crystallization from Ethyl Formate

[0032] (a) 1α-Hydroxyvitamin D₂ product (50 mg) to be purified wasdissolved in boiling ethyl formate (1.2 mL, Aldrich) under argonatmosphere, left at room temperature (68° F.) for a few hours (1-3 hrs)and then in a refrigerator (35-45° F.) overnight (8-12 hrs). Theprecipitated crystals were filtered off, washed with a small volume of acold (0° C.) ethyl formate and dried. The yield of crystalline materialwas 38 mg (76%). HPLC profiles of crystals and mother liquor are shownin FIGS. 3a, 3 b.

[0033] (b) These crystals of 1α-hydroxyvitamin D₂ (26.8 mg) wererecrystallized with ethyl formate (0.5 mL) as described in Example 1(a)and the precipitated crystals (20 mg, 78%), m.p. 153-155° C., wereobserved under a microscope (FIGS. 4a, 4 b) and analyzed bystraight-phase HPLC (crystals: FIG. 2b; mother liquors: FIG. 3c),reverse-phase HPLC (Figure 5b), and ¹H NMR (Figures 1 a, 1 b).

EXAMPLE 2

[0034] Crystallization from Binary Solvent System: EthylAcetate-Petroleum Ether

[0035] (a) 1α-Hydroxyvitamin D₂ product (50 mg) to be purified wasdissolved in boiling ethyl acetate (0.5 mL, Burdick&Jackson) under argonatmosphere and petroleum ether (1.5 mL, b.p. 35-60° C.; Aldrich) wasadded. The solution was left at room temperature (68° F.) for a fewhours (1-3 hrs) and then in a refrigerator (35-45° F.) overnight (8-12hrs). The precipitated crystals were filtered off, washed with a smallvolume of petroleum ether and dried. The yield of crystalline materialwas 32.5 mg (65%). HPLC profiles of crystals and mother liquor are shownin FIGS. 3d, 3 e.

[0036] (b) These crystals of 1α-hydroxyvitamin D₂ (24.8 mg) wererecrystallized with ethyl acetate (0.23 mL) and petroleum ether (0.69mL) as described in Example 2(a) and the precipitated crystals (17 mg,69%), m.p. 149.5-152.50C, were observed under a microscope (FIGS. 4c, 4d) and analyzed by straight-phase HPLC (crystals: FIG. 2c; motherliquors: FIG. 3f, reverse-phase HPLC (FIG. 5c), and ¹H NMR (Figures 1 c,1 d).

EXAMPLE 3

[0037] Crystallization from Ternary Solvent System:2-Propanol-Hexane-Petroleum Ether

[0038] (a) 1α-Hydroxyvitamin D₂ product (50 mg) to be purified wasdissolved in boiling 2-propanol-hexane mixture (15:85; 0.6 mL;Burdick&Jackson) under argon atmosphere and petroleum ether (1.7 mL,b.p. 35-60° C.; Aldrich) was added. The solution was left at roomtemperature (68° F.) for a few hours (1-3 hrs) and then in arefrigerator (35-45° F.) overnight (8-12 hrs). The precipitated crystalswere filtered off, washed with a small volume of petroleum ether anddried. The yield of crystalline material was 34.5 mg (69%). HPLCprofiles of crystals and mother liquor are shown in FIGS. 3g, 3 h.

[0039] (b) These crystals of 1α-hydroxyvitamin D₂ (23.6 mg) wererecrystallized with 2-propanol-hexane mixture (15:85; 0.15 mL) andpetroleum ether (0.4 mL) as described in Example 3(a) and theprecipitated crystals (15.6 mg, 66%), m.p. 154-156° C., were observedunder a microscope (FIGS. 4e, 4 f) and analyzed by straight-phase HPLC(crystals: FIG. 2d; mother liquors: FIG. 3i), reverse-phase HPLC (FIG.5d), and ¹H NMR (Figures 1 e, 1 f).

EXAMPLE 4

[0040] Experimental

[0041] A colorless prism-shaped crystal of dimensions 0.52×0.44×0.38 mmwas selected and designated as 98247 (crystal form I) for structuralanalysis. Intensity data for this compound were collected using a BrukerSMART ccd area detector; (a) Data Collection: SMART Software ReferenceManual (1994). Bruker-AXS, 6300 Enterprise Dr., Madison, Wis.53719-1173, USA; (b) Data Reduction: SAINT Software Reference Manual(1995). Bruker-AXS, 6300 Enterprise Dr., Madison, WI 53719-1173, USA;mounted on a Bruker P4 goniometer using with graphite-monochromated MoKα radiation (λ0.71073 Å). The sample was cooled to 138° K. Theintensity data, which nominally covered one and a half hemispheres ofreciprocal space, were measured as a series of φ oscillation frames-eachof 0.4° for 30 sec/frame. The detector was operated in 512×512 mode andwas positioned 5.00 cm from the sample. Coverage of unique data was98.9% complete to 25.00 degrees in θ. Cell parameters were determinedfrom a non-linear least squares fit of 3054 peaks in the range3.0<θ<25.0°. The first 50 frames were repeated at the end of datacollection and yielded a total of 140 peaks showing a variation of−0.15% during the data collection. A total of 6364 data were measured inthe range 1.96<θ<28.20°. The data were corrected for absorption by theempirical method, G. M. Sheldrick (1996), SADABS, Program for EmpiricalAbsorption Correction of Area Detector Data, University of Göttingen,Germany, giving minimum and maximum transmission factors of 0.744 and0.970. The data were merged to form a set of 4597 independent data withR(int)=0.0320.

[0042] The Monoclinic space group C2 was determined by systematicabsences and statistical tests and verified by subsequent refinement.The structure was solved by direct methods and refined by full-matrixleast-squares methods on F², (a) G. M. Sheldrick (1994), SHELXTL Version5 Reference Manual. Bruker-AXS, 6300 Enterprise Dr., Madison, Wis.53719-1173, USA; (b) International Tables for Crystallography, Vol C,Tables 6.1.1.4, 4.2.6.8, and 4.2.4.2, Kluwer: Boston (1995). Hydrogenatom positions were initially determined by geometry and refined by ariding model. Non-hydrogen atoms were refined with anisotropicdisplacement parameters. A total of 281 parameters were refined against3 restraints and 4597 data to give wR(F²)=0.1311 and S =0.938 forweights of w=1/[σ² (F²)+(0.0734 P)²], where P=[F_(o) ²+2F_(c) ²] /3. Thefinal R(P) was 0.0522 for the 3133 observed, [F>4σ(F)], data. Thelargest shift/s.u. was 0.001 in the final refinement cycle. The finaldifference map had maxima and minima of 0.317 and −0.295 e/Å³,respectively. The absolute structure was determined by refinement of theFlack parameter, H. D. Flack, Acta Cryst. A39, 876-881 (1983). The polaraxis restraints were taken from Flack and Schwarzenbach, H. D. Flack andD. Schwarzenbach, Acta Cryst. A44, 499-506 (1988).

[0043] The displacement ellipsoids were drawn at the 50% probabilitylevel. Methyl group C(2) was disordered and modeled in two orientationswith occupancies of 0.661(9) for the unprimed atom and 0.339(9) for theprimed atom. Restraints were applied to the positional parameters ofthese atoms.

[0044] The three dimensional structure of 1α-hydroxyvitamin D₂ asdefined by the following physical data and atomic positional parametersdescribed and calculated herein is illustrated in FIGS. 6a and 6 b.TABLE 1 Crystal data and structure refinement for 98247 (crystal formI). Identification code 98247 (Form I) Empirical formula C28 H45 O2Formula weight 413.64 Crystal system Monoclinic Space group C2 Unit celldimensions a = 23.952(4) Å α = 90° b = 6.8121(9) Å β = 119.579(2)° c =17.994(2) Å γ = 90° Volume 2553.3(6) Å³ Z 4 Density (calculated) 1.076Mg/m³ Wavelength 0.71073 Å Temperature 138(2) K F(000) 916 Absorptioncoefficient 0.065 mm⁻¹ Absorption correction Empirical Max. and min.transmission 0.970 and 0.744 Theta range for data collection 1.96 to28.20°. Reflections collected 6364 Independent reflections 4597 [R(int)= 0.0320] Data/restraints/parameters 4597/3/281 wR(F² all data) wR2 =0.1311 R(F obsd data) R1 = 0.0522 Goodness-of-fit on F² 0.938 Observeddata [I > 2σ(I)] 3133 Absolute structure parameter 1.2(19) Largest andmean shift/s.u. 0.001 and 0.000 Largest diff. peak and hole 0.317 and−0.295 e/Å³

[0045] TABLE 2 Atomic coordinates and equivalent isotropic displacementparameters for 98247. U(eq) is defined as one third of the trace of theorthogonalized U_(ij) tensor. x y z U(eq) O(1) 0.54852(13) 0.4364(3)0.97881(18) 0.0713(8) O(2) 0.56423(10) 1.0480(3) 1.00431(14) 0.0465(5)C(l) 0.57345(13) 0.6257(4) 0.98084(19) 0.0355(6) C(2) 0.59343(13)0.7273(4) 1.06595(17) 0.0350(7) C(3) 0.61677(13) 0.9353(4) 1.06655(17)0.0349(6) C(4) 0.67265(13) 0.9337(4) 1.04799(16) 0.0327(6) C(5)0.65759(12) 0.8180(4) 0.96806(16) 0.0281(6) C(6) 0.66835(12) 0.8929(4)0.90804(16) 0.0278(6) C(7) 0.64998(12) 0.8085(4) 0.82523(16) 0.0290(6)C(8) 0.66086(12) 0.8835(4) 0.76415(16) 0.0282(6) C(9) 0.69983(14)1.0672(4) 0.77547(18) 0.0364(7) C(10) 0.62870(12) 0.6216(4) 0.96228(17)0.0303(6) C(11) 0.74852(13) 1.0392(4) 0.74434(17) 0.0343(6) C(12)0.71843(13) 0.9532(4) 0.65447(16) 0.0303(6) C(13) 0.68376(11) 0.7601(4)0.64784(15) 0.0266(6) C(14) 0.63338(12) 0.8017(4) 0.67657(16) 0.0301(6)C(15) 0.59228(13) 0.6150(5) 0.65149(17) 0.0385(7) C(16) 0.59231(14)0.5432(5) 0.57008(18) 0.0461(8) C(17) 0.63841(12) 0.6810(4) 0.55701(16)0.0307(6) C(18) 0.73112(12) 0.6019(4) 0.70394(17) 0.0343(6) C(19)0.64831(14) 0.4570(4) 0.94245(18) 0.0391(7) C(20) 0.66588(13) 0.5767(4)0.50566(17) 0.0346(6) C(21) 0.71140(13) 0.7081(4) 0.49045(19) 0.0408(7)C(22) 0.61101(13) 0.5120(4) 0.42223(17) 0.0366(7) C(23) 0.59363(14)0.3296(5) 0.39507(19) 0.0430(7) C(24) 0.53471(15) 0.2714(5) 0.3132(2)0.0515(9) C(25) 0.5488(2) 0.1850(5) 0.2454(3) 0.0788(13) C(26) 0.5847(2)0.3260(6) 0.2199(2) 0.0744(12) C(27) 0.5811(3) −0.0047(7) 0.2677(3)0.055(2) C(27′) 0.5146(6) −0.0003(16) 0.2045(8) 0.110(7) C(28)0.49294(18) 0.1351(6) 0.3345(3) 0.0771(12)

[0046] TABLE 3 Bond lengths [Å] and angles [°] for 98247. O(1)-C(1)1.414(3) C(10)-C(1)-C(2) 110.7(2) O(2)-C(3) 1.427(3) C(3)-C(2)-C(1)111.2(2) C(1)-C(10) 1.517(3) O(2)-C(3)-C(2) 108.5(2) C(1)-C(2) 1.526(4)O(2)-C(3)-C(4) 111.1(2) C(2)-C(3) 1.522(4) C(2)-C(3)-C(4) 110.4(2)C(3)-C(4) 1.531(4) C(5)-C(4)-C(3) 112.7(2) C(4)-C(5) 1.519(4)C(6)-C(5)-C(10) 124.0(2) C(5)-C(6) 1.330(3) C(6)-C(5)-C(4) 121.3(3)C(5)-C(10) 1.486(4) C(10)-C(5)-C(4) 114.7(2) C(6)-C(7) 1.448(3)C(5)-C(6)-C(7) 126.9(2) C(7)-C(8) 1.349(3) C(8)-C(7)-C(6) 127.3(2)C(8)-C(14) 1.484(3) C(7)-C(8)-C(14) 124.1(2) C(8)-C(9) 1.514(4)C(7)-C(8)-C(9) 123.9(2) C(9)-C(11) 1.535(4) C(14)-C(8)-C(9) 111.9(2)C(10)-C(19) 1.331(4) C(8)-C(9)-C(11) 112.2(2) C(11)-C(12) 1.525(3)C(19)-C(10)-C(5) 124.3(2) C(12)-C(13) 1.529(3) C(19)-C(10)-C(1) 122.4(2)C(13)-C(18) 1.529(3) C(5)-C(10)-C(1) 113.3(2) C(13)-C(17) 1.546(3)C(12)-C(11)-C(9) 112.8(2) C(13)-C(14) 1.556(3) C(11)-C(12)-C(13)111.6(2) C(14)-C(15) 1.534(4) C(12)-C(13)-C(18) 111.0(2) C(15)-C(16)1.545(4) C(12)-C(13)-C(17) 117.0(2) C(16)-C(17) 1.553(4)C(18)-C(13)-C(17) 111.0(2) C(17)-C(20) 1.546(3) C(12)-C(13)-C(14)107.3(2) C(20)-C(22) 1.493(4) C(18)-C(13)-C(14) 110.7(2) C(20)-C(21)1.536(4) C(17)-C(13)-C(14) 99.18(18) C(22)-C(23) 1.325(4)C(8)-C(14)-C(15) 120.5(2) C(23)-C(24) 1.505(4) C(8)-C(14)-C(13) 114.3(2)C(24)-C(25) 1.534(4) C(15)-C(14)-C(13) 104.0(2) C(24)-C(28) 1.545(4)C(14)-C(15)-C(16) 104.0(2) C(25)-G(27) 1.457(4) C(15)-C(16)-C(17)106.6(2) C(25)-C(27′) 1.486(6) C(13)-C(17)-C(20) 120.5(2) C(25)-C(26)1.505(5) C(13)-C(17)-C(16) 103.4(2) O(1)-C(1)-C(10) 112.4(2)C(20)-C(17)-C(16) 110.9(2) O(1)-C(1)-C(2) 111.0(2) C(22)-C(20)-C(21)110.1(2) C(22)-C(20)-C(17) 108.3(2) C(27)-C(25)-C(27′) 58.1(6)C(21)-C(20)-C(17) 112.8(2) C(27)-C(25)-C(26) 110.1(4) C(23)-C(22)-C(20)127.5(3) C(27′)-C(25)-C(26) 130.4(6) C(22)-C(23)-C(24) 125.4(3)C(27)-C(25)-C(24) 114.6(4) C(23)-C(24)-C(25) 114.3(3) C(27′)-C(25)-C(24)115.9(5) C(23)-C(24)-C(28) 108.9(3) C(26)-C(25)-C(24) 112.4(3)C(25)-C(24)-C(28) 112.9(3)

[0047] TABLE 4 Anisotropic displacement parameters (Å² × 10³) for 98247.The anisotropic displacement factor exponent takes the form:−2π²[h²a^(*2)U₁₁ + . . . + 2hka^(*)b^(*)U₁₂] U₁₁ U₂₂ U₃₃ U₂₃ U₁₃ U₁₂O(1) 123(2)  19(1) 138(2)  −9(1)− 115(2)  −12(1)  O(2) 58(1) 34(1) 68(1)17(1)  46(1) 21(1)  C(1) 49(2) 17(1) 61(2) 4(1) 42(2) 2(1) C(2) 42(2)30(2) 47(2) 12(1)  33(1) 9(1) C(3) 45(2) 32(2) 39(2) 6(1) 28(1) 7(1)C(4) 39(2) 31(2) 36(1) 5(1) 24(1) 4(1) C(5) 29(1) 24(1) 34(1) 4(1) 18(1)6(1) C(6) 25(1) 25(1) 35(1) 1(1) 17(1) 0(1) C(7) 28(1) 26(1) 35(1) 2(1)16(1) 0(1) C(8) 27(1) 25(1) 34(1) 2(1) 16(1) 2(1) C(9) 45(2) 30(2) 40(2)−1(1)  25(1) −8(1)  C(10) 38(2) 21(1) 41(2) 9(1) 27(1) 8(1) C(11) 41(2)28(2) 40(2) 0(1) 25(1) −9(1)  C(12) 33(1) 29(1) 33(1) 5(1) 20(1) 2(1)C(13) 23(1) 28(2) 32(1) 5(1) 16(1) 2(1) C(14) 26(1) 33(2) 33(1) 3(1)16(1) 1(1) C(15) 30(1) 50(2) 42(2) −13(1)  23(1) −12(1)  C(16) 36(2)62(2) 46(2) −15(2)  25(1) −13(2)  C(17) 25(1) 38(2) 31(1) −2(1)  15(1)1(1) C(18) 35(2) 28(2) 43(2) 5(1) 21(1) 2(1) C(19) 45(2) 37(2) 44(2)10(1)  29(2) 9(1) C(20) 37(2) 33(2) 40(2) −3(1)  23(1) −1(1)  C(21)44(2) 41(2) 51(2) −10(1)  34(2) −7(1)  C(22) 43(2) 32(2) 42(2) −1(1) 26(1) −1(1)  C(23) 46(2) 42(2) 53(2) −5(2)  34(2) −4(2)  C(24) 55(2)52(2) 65(2) −31(2)  43(2) −24(2)  C(25) 103(3)  67(3) 105(3)  −42(2) 81(3) −38(2)  C(26) 100(3)  76(3) 82(3) −27(2)  71(2) −28(2)  C(27)84(4) 43(3) 61(4) 1(2) 54(3) 6(3) C(27′) 87(11) 166(17)  81(10) −77(10) 45(9) −31(10)  C(28) 77(3) 79(3) 106(3)  −40(2)  70(2) −40(2) 

[0048] TABLE 5 Hydrogen coordinates and isotropic displacementparameters for 98247. x y z U(eq) H(1A) 0.5187 0.4364 0.9914 0.107 H(1B)0.5682 0.3243 1.0063 0.107 H(2A) 0.5737 1.1693 0.9770 0.070 H(2B) 0.53191.0320 1.0104 0.070 H(1) 0.5382 0.7054 0.9350 0.043 H(2A) 0.5564 0.73141.0761 0.042 H(2B) 0.6281 0.6510 1.1128 0.042 H(3) 0.6318 0.9944 1.12420.042 H(4A) 0.6833 1.0705 1.0412 0.039 H(4B) 0.7108 0.8758 1.0974 0.039H(6) 0.6904 1.0149 0.9208 0.033 H(7) 0.6278 0.6868 0.8125 0.035 H(9A)0.7230 1.1039 0.8367 0.044 H(9B) 0.6704 1.1762 0.7433 0.044 H(11A)0.7681 1.1676 0.7450 0.041 H(11B) 0.7832 0.9509 0.7844 0.041 H(12A)0.7524 0.9301 0.6393 0.036 H(12B) 0.6875 1.0486 0.6132 0.036 H(14)0.6048 0.9068 0.6375 0.036 H(15A) 0.5481 0.6445 0.6395 0.046 H(15B)0.6114 0.5154 0.6974 0.046 H(16A) 0.5485 0.5500 0.5202 0.055 H(16B)0.6075 0.4056 0.5772 0.055 H(17) 0.6123 0.7945 0.5218 0.037 H(18A)0.7505 0.6404 0.7641 0.051 H(18B) 0.7084 0.4770 0.6951 0.051 H(18C)0.7648 0.5868 0.6885 0.051 H(19A) 0.6649 0.3664 0.9910 0.059 H(19B)0.6121 0.3958 0.8927 0.059 H(19C) 0.6823 0.4877 0.9290 0.059 H(20)0.6901 0.4578 0.5382 0.042 H(21A) 0.7217 0.6437 0.4500 0.061 H(21B)0.6905 0.8343 0.4669 0.061 H(21C) 0.7510 0.7300 0.5447 0.061 H(22)0.5856 0.6132 0.3842 0.044 H(23) 0.6206 0.2269 0.4302 0.052 H(24) 0.50930.3940 0.2883 0.062 H(25) 0.5062 0.1628 0.1932 0.095 H(25′) 0.58690.1178 0.2864 0.095 H(26A) 0.5891 0.2695 0.1730 0.112 H(26B) 0.56110.4500 0.2013 0.112 H(26C) 0.6274 0.3502 0.2689 0.112 H(27A) 0.5881−0.0496 0.2212 0.083 H(27B) 0.6225 0.0083 0.3202 0.083 H(27C) 0.5544−0.1002 0.2767 0.083 H(27D) 0.5271 −0.0434 0.1627 0.164 H(27E) 0.5261−0.1018 0.2482 0.164 H(27F) 0.4682 0.0222 0.1754 0.164 H(28A) 0.47770.2077 0.3681 0.116 H(28B) 0.4561 0.0889 0.2813 0.116 H(28C) 0.51860.0224 0.3676 0.116

[0049] TABLE 6 Torsion angles [°] for 98247. O(1)-C(1)-C(2)-C(3)−176.8(2) C(12)-C(13)-C(14)-C(8) −57.5(3) C(10)-C(1)-C(2)-C(3) 57.6(3)C(18)-C(13)-C(14)-C(8) 63.7(3) C(1)-C(2)-C(3)-O(2) 64.8(3)C(17)-C(13)-C(14)-C(8) −179.7(2) C(1)-C(2)-C(3)-C(4) −57.2(3)C(12)-C(13)-C(14)-C(15) 169.2(2) O(2)-C(3)-C(4)-C(5) −69.0(3)C(18)-C(13)-C(14)-C(15) −69.6(2) C(2)-C(3)-C(4)-C(5) 51.4(3)C(17)-C(13)-C(14)-C(15) 47.0(2) C(3)-C(4)-C(5)-C(6) 130.8(3)C(8)-C(14)-C(15)-C(16) −162.1(2) C(3)-C(4)-C(5)-C(10) −47.5(3)C(13)-C(14)-C(15)-C(16) −32.4(3) C(10)-C(5)-C(6)-C(7) 5.3(4)C(14)-C(15)-C(16)-C(17) 5.0(3) C(4)-C(5)-C(6)-C(7) −172.8(2)C(12)-C(13)-C(17)-C(20) 77.7(3) C(5)-C(6)-C(7)-C(8) −179.8(3)C(18)-C(13)-C(17)-C(20) −51.0(3) C(6)-C(7)-C(8)-C(14) −171.8(2)C(14)-C(13)-C(17)-C(20) −167.4(2) C(6)-C(7)-C(8)-C(9) 4.9(4)C(12)-C(13)-C(17)-C(16) −157.8(2) C(7)-C(8)-C(9)-C(11) 133.3(3)C(18)-C(13)-C(17)-C(16) 73.5(3) C(14)-C(8)-C(9)-C(11) −49.6(3)C(14)-C(13)-C(17)-C(16) −42.9(3) C(6)-C(5)-C(10)-C(19) 49.5(4)C(15)-C(16)-C(17)-C(13) 24.2(3) C(4)-C(5)-C(10)-C(19) −132.3(3)C(15)-C(16)-C(17)-C(20) 154.8(2) C(6)-C(5)-C(10)-C(1) −130.1(3)C(13)-C(17)-C(20)-C(22) 178.3(2) C(4)-C(5)-C(10)-C(1) 48.1(3)C(16)-C(17)-C(20)-C(22) 57.5(3) O(1)-C(1)-C(10)-C(19) 3.0(4)C(13)-C(17)-C(20)-C(21) −59.6(3) C(2)-C(1)-C(10)-C(19) 127.8(3)C(16)-C(17)-C(20)-C(21) 179.5(2) O(1)-C(1)-C(10)-C(5) −177.3(2)C(21)-C(20)-C(22)-C(23) 121.8(3) C(2)-C(1)-C(10)-C(5) −52.5(3)C(17)-C(20)-C(22)-C(23) −114.4(3) C(8)-C(9)-C(11)-C(12) 50.4(3)C(20)-C(22)-C(23)-C(24) 174.8(3) C(9)-C(11)-C(12)-C(13) −55.2(3)C(22)-C(23)-C(24)-C(25) 110.7(4) C(11)-C(12)-C(13)-C(18) −64.9(3)C(22)-C(23)-C(24)-C(28) −121.9(3) C(11)-C(12)-C(13)-C(17) 166.5(2)C(23)-C(24)-C(25)-C(27) 67.2(4) C(11)-C(12)-C(13)-C(14) 56.2(3)C(28)-C(24)-C(25)-C(27) −58.1(5) C(7)-C(8)-C(14)-C(15) −3.1(4)C(23)-C(24)-C(25)-C(27′) 132.1(8) C(9)-C(8)-C(14)-C(15) 179.9(2)C(28)-C(24)-C(25)-C(27′) 6.8(8) C(7)-C(8)-C(14)-C(13) −128.0(3)C(23)-C(24)-C(25)-C(26) −59.5(5) C(9)-C(8)-C(14)-C(13) 54.9(3)C(28)-C(24)-C(25)-C(26) 175.2(4)

[0050] TABLE 7 Hydrogen bonds for 98247[Å and °]. D-H . . . A d(D-H) d(H. . . A) d(D . . . A) <(DHA) O(1)-H(1A) . . . O(1)#1 0.85 1.93 2.781(4)179.4 O(1)-H(1B) . . . O(2)#2 0.91 1.88 2.679(3) 145.5 O(2)-H(2A) . . .O(1)#3 1.04 1.92 2.679(3) 126.8 O(2)-H(2B) . . . O(2)#1 0.84 2.193.004(4) 163.7

[0051] Symmetry transformations used to generate equivalent atoms:#1−x+1, y, −z+2 #2 x, y−1, z #3 x, y+1, z

EXAMPLE 5

[0052] Experimental

[0053] From the crystals recovered in Example 2, a second colorlessneedle-shaped crystal of dimensions 0.4×0.05×0.05 mm was selected anddesignated as crystal form II for structural analysis. Data werecollected in the same manner as set forth in Example 4, and is reportedin Table 8. The data reported in Tables 2-7 herein are also applicableto crystal form II. Although the 1α-hydroxyvitamin D₂ crystal recoveredhas a molecular packing arrangement defined by space group C2, and theunit cell dimensions in Table 8, the crystal could also be defined byany other space group that yields substantially the same crystallinepacking arrangement. TABLE 8 Crystal data and structure refinement forcrystal form II. Identification code Form II Empirical formula C28 H45O2 Formula weight 413.64 Crystal system Monoclinic Space group C2 Unitcell dimensions a = 23.9(1) Å α = 90° b = 6.8(1) Å β = 100.9(2)° c =31.7(1) Å γ = 90° Volume 2553.3(6) Å³ Z 4 Density (calculated) 1.076Mg/m³ Wavelength 0.71073 Å Temperature 138(2) K F(000) 916 Absorptioncoefficient 0.065 mm⁻¹ Absorption correction Empirical Max. and min.transmission 0.970 and 0.744 Theta range for data collection 1.96 to28.20°. Reflections collected 6364 Independent reflections 4597 [R(int)= 0.0320] Data/restraints/parameters 4597/3/281 wR(F²all data) wR2 =0.1311 R(F obsd data) R1 = 0.0522 Goodness-of-fit on F² 0.938 Observeddata [I > 2σ(I)] 3133 Absolute structure parameter 1.2(19) Largest andmean shift/s.u. 0.001 and 0.000 Largest diff. peak and hole 0.317 and−0.295 e/Å³

EXAMPLE 6

[0054] Experimental

[0055] From the crystals recovered in Example 3, a third colorlessplate-shaped crystal of dimensions 0.4×0.15×0.05 mm was selected anddesignated as crystal form III for structural analysis. Data werecollected in the same manner as set forth in Example 4, and is reportedin Table 9. The data reported in Tables 2-7 herein is also applicable tocrystal form III. Although the 1α-hydroxyvitamin D₂ crystal recoveredhas a molecular packing arrangement defined by space group C2, and theunit cell dimensions in Table 9, the crystal could also be defined byany other space group that yields substantially the same crystallinepacking arrangement. TABLE 9 Crystal data and structure refinement forcrystal form III. Identification code Form III Empirical formula C28 H45O2 Formula weight 413.64 Crystal system Monoclinic Space group C2 Unitcell dimensions a = 28.5(1) Å α= 90° b = 6.7(1) Å β= 105.6(2)° c =28.2(1) Å γ= 90° Volume 2553.3(6) Å³ Z 4 Density (calculated) 1.076Mg/m³ Wavelength 0.71073 Å Temperature 138(2) K F(000) 916 Absorptioncoefficient 0.065 mm⁻¹ Absorption correction Empirical Max. and min.transmission 0.970 and 0.744 Theta range for data collection 1.96 to28.20°. Reflections collected 6364 Independent reflections 4597 [R(int)= 0.0320] Data/restraints/parameters 4597/3/281 wR(F² all data) wR2 =0.1311 R(F obsd data) R1 = 0.0522 Goodness-of-fit on F² 0.938 Observeddata [I > 2σ(I)] 3133 Absolute structure parameter 1.2(19) Largest andmean shift/s.u. 0.001 and 0.000 Largest diff. peak and hole 0.317 and−0.295 e/Å³

I claim:
 1. 1α-hydroxyvitamin D₂ in crystalline form having a molecularpacking arrangement defined by space group C2 and unit cell dimensionsa=23.9A°, b=6.8A°, c=31.7A°, α=90°, β=100.9° and γ=90°, or any otherspace group that yields substantially the same crystalline packingarrangement.
 2. 1α-hydroxyvitamin D₂ in crystalline form having amolecular packing arrangement defined by space group C2 and unit celldimensions a=28.5A°, b=6.7A°, c=28.2A°, α=90°, β=105.6° and γ=90°, orany other space group that yields substantially the same crystallinepacking arrangement.
 3. 1α-hydroxyvitamin D₂ in crystalline form havinga melting point of about 149.5° C. to about 152.5° C. and having amolecular packing arrangement defined by space group C2 and unit celldimensions a=23.9A°, b=6.8A°, c=31.7A°, α=90°, β=100.9° and γ=90°, orany other space group that yields substantially the same crystallinepacking arrangement.
 4. 1α-hydroxyvitamin D₂ in crystalline form havinga melting point of about 154° C. to about 156° C. and having a molecularpacking arrangement defined by space group C2 and unit cell dimensionsa=28.5A°, b=6.7A°, c=28.2A°, α=90°, β=105.6° and y=90°, or any otherspace group that yields substantially the same crystalline packingarrangement.
 5. A method of purifying 1α-hydroxyvitamin D_(2,)comprising the steps of: (a) boiling a solvent comprising ethyl acetateunder inert atmosphere; (b) dissolving a product containing1α-hydroxyvitamin D₂ to be purified in said solvent; (c) addingpetroleum ether to said solvent after dissolving said product in saidsolvent; (d) cooling said solvent and dissolved product below ambienttemperature for a sufficient amount of time to form a precipitate of1α-hydroxyvitamin D₂ crystals; and (e) recovering the 1α-hydroxyvitaminD₂ crystals having a molecular packing arrangement defined by spacegroup C2 and unit cell dimensions a=23.9A°, b=6.8A°, c=31.7A°, α=90°,P=100.9° and y=90°, or any other space group that yields substantiallythe same crystalline packing arrangement.
 6. The method of claim 5further including the step of allowing said solvent and dissolvedproduct to cool to ambient temperature prior to cooling below ambienttemperature.
 7. The method of claim 5 wherein said inert atmosphere isan argon atmosphere.
 8. The method of claim 5 wherein said solvent anddissolved product is cooled to between about 35° F. to about 45° F. 9.The method of claim 5 wherein the step of recovering comprisesfiltering.
 10. The method of claim 5 further including the step of (irepeating steps (a) through (e) using the recovered crystals from step(e) as the product of step (b).
 11. A method of purifying1α-hydroxyvitamin D_(2,) comprising the steps of: (a) boiling a solventcomprising 2-propanol-hexane mixture under inert atmosphere; (b)dissolving a product containing 1α-hydroxyvitamin D₂ to be purified insaid solvent; (c) adding petroleum ether to said solvent afterdissolving said product in said solvent; (d) cooling said solvent anddissolved product below ambient temperature for a sufficient amount oftime to form a precipitate of 1α-hydroxyvitamin D₂ crystals; and (e)recovering the 1α-hydroxyvitamin D₂ crystals having a molecular packingarrangement defined by space group C2 and unit cell dimensions a=28.5A°,b=6.7A°, c=28.2A°, α=90°, β=105.6 ° and γ=90°, or any other space groupthat yields substantially the same crystalline packing arrangement. 12.The method of claim 11 further including the step of allowing saidsolvent and dissolved product to cool to ambient temperature prior tocooling below ambient temperature.
 13. The method of claim 11 whereinsaid inert atmosphere is an argon atmosphere.
 14. The method of claim 11wherein said solvent and dissolved product is cooled to between about35° F. to about 45° F.
 15. The method of claim 11 wherein the step ofrecovering comprises filtering.
 16. The method of claim 11 furtherincluding the step of (f repeating steps (a) through (e) using therecovered crystals from step (e) as the product of step (b).